U.S. patent number 3,678,151 [Application Number 04/845,089] was granted by the patent office on 1972-07-18 for biological staining method.
This patent grant is currently assigned to Gugol-Clini-Tex, Inc.. Invention is credited to Andrew Horonick, Andre V. Munschy.
United States Patent |
3,678,151 |
Horonick , et al. |
July 18, 1972 |
BIOLOGICAL STAINING METHOD
Abstract
The invention relates to preparation of precise micro and other
values of volumetric concentrations of chemicals, standards,
reagents, pharmaceuticals and biologicals in solvents therefor and
utilizes dry segments from an inert, integrally divisible and
absorbent carrier uniformly impregnated with said substances, each
of the segments carrying a computed quantity of the impregnation
based on the proportionate size with respect to the carrier. One of
the objects of the invention is in the preparation and utilization
of segments as dry stain or dye strips for formation of liquid
stains applicable to biological materials such as blood smears and
bacteria for staining and microscopic examination purposes.
Inventors: |
Horonick; Andrew (Bronx,
NY), Munschy; Andre V. (Bronx, NY) |
Assignee: |
Gugol-Clini-Tex, Inc. (New
York, NY)
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Family
ID: |
25294360 |
Appl.
No.: |
04/845,089 |
Filed: |
July 25, 1969 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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562487 |
Jul 5, 1966 |
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Current U.S.
Class: |
435/40.5; 435/29;
436/169; 435/808; 435/40.51 |
Current CPC
Class: |
G01N
1/30 (20130101); Y10S 435/808 (20130101) |
Current International
Class: |
G01N
1/30 (20060101); G01n 001/30 (); G01n 033/16 () |
Field of
Search: |
;424/3,7 ;252/408 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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831,804 |
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Mar 1960 |
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GB |
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135,829 |
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Oct 1902 |
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DD |
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Other References
Lillie, Histopath. Tech. & Practical Histochem. The Blakiston
Div., McGraw-Hill, N.Y., 1965, pp. 585-588..
|
Primary Examiner: Meyers; Albert T.
Assistant Examiner: Fagelson; Anna P.
Parent Case Text
The invention herein is a continuation-in-part of our invention in
our copending application U.S. Ser. No. 562,487 filed July 5, 1966,
now abandoned.
Claims
We claim:
1. A method of staining a biological specimen on a slide for
microscopic examination using a microbiological dye-staining strip
system which comprises,
a. providing a first dry strip of uniformly dimensioned inert
fibrous material uniformly impregnated with a predetermined amount
of dye-stain selective to said specimen,
b. wetting said strip with a catalyst comprising an alcoholic
solution and applying said strip to said specimen thereby
transferring the dye thereto to produce the desired stain, the
catalyst solution being selective to said stain being employed to
augment the stainability of said specimen,
c. removing the strip from the stained specimen,
d. providing another dry strip of uniformly dimensioned inert
fibrous material uniformly impregnated with a predetermined amount
of a soluble buffer material selective to said stain produced by
said first strip, and
e. causing the buffer solution produced from said second strip to
contact the stained specimen.
2. The method of claim 1, wherein the dye-stain in the first dry
strip is a Wright's Stain.
Description
This invention relates generally to premeasured biological
standards, reagents, controls, chemicals and pharmaceuticals
including organic compositions and dyes; and has for objects
procedure for quantitative preparation thereof by impregnation in
and subdividing absorbing carriers therefor.
More particularly, the invention has as an object the dispensing
quantitatively of micro, trace, and other amounts of chemicals,
standards, reagents, medicines, pharmaceuticals, dyes, and the like
by incorporating said substances (when capable of being reduced to
dry form) in inert carriers and then subdividing the carriers
whereby computable quantities of said substances are dispensable by
the subdivisions.
As to standards, reagents, chemicals, dyes and controls as prepared
in clinical laboratories, they are customarily provided for use in
liquid form and are dispensed in accurately measured volumes.
However, such compositions are not stable for long periods of time,
must be refrigerated or else continually prepared afresh before
use.
The known method used to overcome such shortcomings of unstable
standards, reagents, dyes and controls is to package these products
in powder form accurately weighed out, and then before use to be
diluted to fluid form as is the procedure with lyophilized control
serums and thromboplasteins which are reconstituted. This method,
however, involves a high processing cost and a high margin of
wastage; for when the powder is dissolved and diluted, only a small
portion is used immediately and the remainder spoils and is no
longer useable within a day or two.
The invention has a particular application to quantitative form,
utilization and administration of medicines, drugs, and dyes.
As a further object, the invention embraces a composition of matter
comprising a segment of an accurately divisible, inert, and
absorbent carrier and containing a deposition of one or more
substances in dry from, the quantity of said deposition being a
function of the dimensional division of the carrier.
To overcome present shortcomings in the preparation and use of
chemicals including biologicals and dyes, which by nature are
unstable or which involve difficulties both physical and economic
in preparation and application, the invention has an object a
procedure for and product formation therefrom for increasing the
stability and ease of application of said chemicals to be diluted
as by (1) having distributed in an absorbent inert carrier
premeasured quantities of the selected substance or substance in
dry form; (2) cutting said carrier mathematically into divisions to
arrive at premeasured quantities in each such division; and (3)
placing the carrier division or divisions in conventional
applicable diluents for the active ingredients such as water,
saline, organic solvent, blood serum and whole blood and other
biological fluids. Within a brief time, a definite solution with a
defined concentration is ready for use. Among the agents involved
are serum proteins, hemoglobin and hematin, heparin, pyruvic acid,
glucose, urea, ascorbic acid, carotene, fibrogen, vitamin A or
vitamin combinations, calcium, chlorides, cholesterol, and
cholesterol esters, creatinine, phosphorous, lipase, amylase, acid
and alkaline phosphatase, serum trausamines, etc. Agents such as
carotene, vitamin A and cholesterol require organic diluents.
To overcome shortcomings of present preparations for consumption in
premeasured and ingestable dry form, the same procedure is followed
as set forth in the preceding paragraph except that the diluent is
afforded by the body's physiology.
The method of preparing premeasured quantities of stable standards
including reagents, controls, dyes for staining biologicals,
indicators, enzymes and the like, and other biologicals and
chemicals used for human consumption in dry form is as follows.
An absorbent inert carrier such as cellulose, cellulosic
derivatives, glass cloth, asbestos cloth, and other fibers is used
as an effective combined vessel and carrier for a required
concentration of one or more of the said substances in solution
form, and the diluent is then evaporated from said carrier.
Thereafter, the carrier having the active ingredients distributed
therein in dry from form may be subdivided mathematically and
concentration-wise into a plurality of forms and shapes or into
compressed tablets depending both upon the particular material of
the carrier and the use to which it is to be put. The user will
thus have available measured micogram and milligram or other
quantities of any agent or substance or combinations in which he is
interested. Moreover, the prepared strips and tablets of said
substances or combinations maintain the chemicals in stable form in
a non-humid environment for an indefinite period of time without
refrigeration requirements; and as to those substances sensitive to
light and other rays, suitable shields therefor in containers may
be provided.
As to the substances requiring liquid dilution before use, one or
more of the impregnated dry carrier units is immersed in a given or
selected quantity of diluent or leaching fluid such as water,
saline, organic solvent, blood serum, whole blood and other
biological fluids for a few minutes for leaching purposes, and then
any test procedure may be begun. Utilization of said units for
staining blood or bacteria in the form of staining strips will
hereinafter be described.
As to the substances for human intake orally in dry form, the
impregnated dry carrier units alone are used but in such cases the
carrier composition should be of ingestable substances without
deleterious effects and even glass cloth and asbestos cloth as
mentioned could be utilized.
When needed for laboratory use in liquid form, the dry carrier
units impregnated with the selected premeasured active ingredient
or ingredients are suspended in a menstrum of diluent such as the
aforestated water, saline, blood serum and other biological fluids
and organic solvents, and after a brief leaching time, the user has
a solution of the substance ready for use and manipulation. This
eliminates measuring out of powders on a balance. For example, if
0.3 mg. of phosphorous in 5 ml. of diluent is required, an
impregnated carrier unit of 0.3 mg. is immersed in 5 ml. of
diluent, and after a brief period of leaching, the user has his
requirements. Moreover, solutions of double, triple and quadruple
concentration can be prepared just as easily merely be increasing
the number of units proportionally.
The carriers preferably are in disc form or cut-outs therefrom
although strip or other forms as mentioned can be used also. Discs
will range in diameter from 5 mm. to 25 mm. and in thickness from
0.005 in. to 0.10 in. The stabilized active ingredients in each
disc can range in amount from traces to necessary amounts.
Combinations of ingredients may vary in number up to 20.
The user will thus have available microgram and milligram
quantities as well as larger quantities exactly measured of any
substance or combination of substances in which he is interested.
Use of the invention in connection with the compositions requiring
leaching will obviate the need to purchase a microbalance and will
save the operator 15 to 30 minutes to a half day of actual working
time in addition to effecting cost economies in use of the
materials; while use of the invention in connection with the
formation of pills, vitamins and other compositions taken orally
together with the carrier and in dry form assures both the user and
manufacturer exact premeasured quantity, stability and freshness
and economy in cost both to manufacturer and consumer.
Below are typical examples for preparation and products of the
invention and are applicable also to compositions used for oral
intake in dry form as aforedescribed.
HEMOGLOBIN STANDARD AND CONTROL
Outdated bank blood was placed in a jar and a strip of filter
paper--pure cellulose--obtained from the Whatman Filter Paper
distributor, Reeves Angel, New Jersey, was slowly drawn through the
well mixed blood. The coated paper was suspended above a hot plate
and the rising warm air dried the strip within an hour.
A paper punch with a 1/4 in. die was used to punch out discs from
the coated strip.
The circles were then suspended in Drabkin's solution (used
generally for hemoglobin determination) and within 15 minutes,
readings were begun on a colorimeter with the following
results.
Tube No. of Discs Reading 1) 1 62 63 63 2) 2 119 122 118 3) 3 180
182 176 4) 4 226 224 220 (11/29/65) (12/1/65) (12/10/65)
GLUCOSE STANDARD AND CONTROL
Three solutions were prepared in 100 ml. of water each as
follows:
1) 90 mg. uric acid 1500 mg. glucose 2) 90 mg. uric acid 1500 mg.
glucose 1000 mg. urea
3) 90 mg. uric acid 1500 mg. glucose 1000 mg. urea 250 mg.
potassium dihydrogen phosphate.
Discs were prepared as before for hemoglobin. Glucose was then
determined using the Folin Wu method. Each disc contains the same
amount of glucose and should give the same readings.
Tube Items Reading 1) 2 Discs Soln 1 232 2) 2 Discs Soln 1 238 3) 2
Discs Soln. 2 230 4) 2 Discs Soln. 2 238 5) 2 Discs Soln 3 230 6) 2
Discs Soln 3 230
The invention herein further encompasses novel staining strips,
preparation and novel utilization thereof in connection with
staining biological specimens such as dried-out blood smears,
heat-fixed bacteria and tissue for microscopic examination on
slides.
Presently, procedure follows a wet process involving the pouring of
a dye over the specimen such as used in applying the Wright's Stain
comprising Methylene Blue and Eosin for microscopic examination of
white blood cells. Thus one pours a proper mixture of liquid dye
and buffer over the specimen. After a wait of about 2 to 4 minutes
for fixation in the case of the said blood smear, an equal volume
of aqueous buffer such as 2 cc of a phosphate buffer is poured over
said specimen at which time staining begins. After a further wait
of 4 to 6 minutes, the excess of dye and buffer is decantered and
the slide is rinsed under tap water or buffer (for maintenance of
necessary pH). Thereafter, the slide is dried under ambient
conditions and is then ready for microscopic examination.
The drawbacks of the procedure above outlined frequently involves
the deterioration of the liquid dye or mixture when same is stored
in a bottle on the shelf such as by formation of formaldehyde and
formic acid from the methyl alcohol solvent. Furthermore
overstaining and understaining may occur because of difficulty in
maintaining proper volume of either dye or stain and buffer or
equal volumes of buffer and dye or stain because of run-off and
spillage from the slide; overstaining and understaining also occur
when the time period of the fixing and buffer stages are not held
within proper limits. If there is undertiming, the specimen is
understained; and if there is overtiming, the specimen is
overstained. With overstaining, a precipitate develops on the
specimen. In either case, differentiability of the the white blood
cells under the microscope is impaired. Under present procedures
there is poor reproduceability of specimens from the same
blood-smear and the operations are messy. As to poor
reproduceability, if the quantitative and timing factors above
described are not maintained in value, the reproduceability of
staining will be variable; and with such different stainings,
different judgments from microscopic examination of similar
specimens will be made. However, in the case of bacteria and tissue
staining, timing is not too important, but the element of messiness
involved in the procedure still applies.
The above shortcomings involved in the staining of biological
specimens are overcome by the structure and application of dry
segments or strips impregnated as mentioned in this specification
but as specifically related to dyes for formation of stain strips
and utilization thereof as will hereinafter appear.
STAINING STRIPS FOR BIOLOGIC SPECIMENS
In the production of typical strips for staining white blood cells
from specimens of blood-smears, we use chemically pure Whatman
filter paper No. 1 in roll or strip form for impregnation with a
mixture found suitable and in following proportions.
Eosin Y 1 gram Methylene Blue 1 gram Azure A 0.6 gram Methylene
Violet 0.2 gram Methyl Alcohol 250 ml.
Above formulation is subject to minor modifications.
A solution of the above ingredients in said methyl alcohol is
prepared in a vessel and the paper is saturated therewith by
passage therethrough. As the paper emerges from the bath it is
suitably dried by evaporation as by passage through a hot air
chamber. Thus, a long strip or roll of dried impregnated paper
results and the impregnation is homogeneously and evenly
distributed throughout.
The impregnated paper is then cut into suitably sized segments such
as seven eighths by 11/2 inches. Since both the total area of the
paper prior to impregnation and the quantity of ingredients of the
saturating solution are known, the quantity of dye in any segmented
piece is arrived at by calculation. Each of the same-sized segments
thus carries the proper, known and equal quantity of dye for
staining purposes. There are no errors and problems due to
spillage. The strips are suitably packaged in boxes and stored and
sold in dry form.
In the utilization of the prepared staining strips, each is
suitably grasped and dipped into a methyl alcohol solution of a
catalyst. The catalyst enables the stain to go into solution in
alcohol. For when the alcohol is evaporated in the formation of the
strips, the paper for some reason affects the function of dye
stainability. Therefore, the alcoholic solution containing the
catalyst restores or augments dye stainability. The catalyst
mixture as used consists of a 1 percent methyl alcohol solution of
glycerol. Alternative mixtures may be a 1 percent methyl alcohol
solution of propylene glycol or methyl alcohol with a trace of
chlorophyl.
The dry strip is immersed into the catalyst alcohol solution for
1-3 seconds until saturated, then withdrawn and placed over the dry
blood-smear on the slide to which said strip adheres for a period
of from 20-90 seconds. Thereafter, the strip is peeled off and
discarded. The slide is then rinsed in a suitable buffer solution
such as a phosphate buffer with pH 6.6-6.8.
After the slide is thus rinsed, it is permitted to dry under
ambient conditions for about 2-4 minutes, and thereafter the slide
is ready for microscopic examination.
The actual staining step according to the above strip procedure
takes between about 20-90 seconds whereas under conventional
procedure of staining, the operation consumes from between 6-10
minutes with the incidental faults of under and over-staining,
variations in reproduceability and messiness of procedure.
Bacteriological stains such as Acid Fast and Gram's Stain and
tissue stains in dry strip form are similarly prepared from methyl
alcohol solutions of dyes. As is known, there are a number of
different dyes or stains used for specific bacteria and tissue
types. Among such dyes or stains available are solutions of 20
percent Basic Fuchsin; 2 percent Crystal Violet; 2 percent
Brilliant Green; 2 percent Methylene Blue; 2 percent Acid Green.
However, there is a wide latitude in formula concentrations.
In the utilization of the bacterial and stain strips, the dry-strip
is placed over the specimen, a few drops of methyl alcohol or water
is added on to the strip causing adherence, and after 1 or 2
minutes, the strip is peeled off, the slide rinsed in tap water,
suitably dried and the stained specimen on the slide is then ready
to be viewed under the microscope.
The phosphate buffer above mentioned and employed in the process
utilizing the dry staining strips to stain the white blood cells
may also be similarly produced in the form of dry strips by
utilizing aqueous 0.2 molar solution of a phosphate, citrate,
tartrate or borate bath. The paper is run therethrough, dried,
segmented and ready for use. Each segment of such prepared strip
submerged in 100 cc of water will yield a buffer solution of the
desired pH.
Thus has been described a novel product, a novel method of
production thereof and novel procedures in utilization in
connection with providing divisions of an accurately divisible,
inert and absorbent carrier containing a uniformly dispersed
deposition of substances in dry form for transfer thereof into
solution for staining and other purposes, the quantity of said
deposition in the divisions being a function of the proportionate
size thereof with respect to the carrier.
* * * * *